Truncation of human squalene synthase yields active, crystallizable protein.
Squalene synthase catalyzes the first committed step in cholesterol biosynthesis and thus is important as a potential target for therapeutic intervention. In order to determine the important functional domains of the protein, the amino and carboxyl terminal regions thought to be involved in membrane association of the enzyme were removed genetically. The 30 N-terminal amino acids were deleted with no apparent effect on activity. Additional deletion of 81 or 97 amino acids from the C-terminus completely ablated activity. However, a protein with a C-terminal deletion of 47 amino acids retained full activity. The latter enzyme was readily overexpressed in Escherichia coli and purified to homogeneity. The pure, doubly truncated enzyme exhibited a specific activity similar to that reported for the protease-solubilized rat liver enzyme, had a KM for farnesyl diphosphate similar to that observed for native enzyme, and was inhibited by anionic compounds to the same degree as native enzyme. Using the vapor diffusion method, the protein was crystallized as an enzyme-inhibitor complex, yielding orthorhombic crystals which diffracted to 2.2 A.
Thompson
J F
JF
Central Research Division, Pfizer Inc., Groton, Connecticut 06340, USA. thompson-jf@pfizer.com
Danley
D E
DE
Mazzalupo
S
S
Milos
P M
PM
Lira
M E
ME
Harwood
H J
HJ
D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000235:genetics; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D012717:Sesquiterpenes
D004791:Enzyme Inhibitors; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D013186:Farnesyl-Diphosphate Farnesyltransferase